The terminal differentiation of epidermal keratinocytes results in the formation of a structure, the stratum corneum, which provides a protective barrier between an organism and its environment. The rapid transition from a living epidermal granular cell to a anucleate cornified squame is regulated by multiple signaling molecules and pathways that are poorly understood. One important group of molecules involved in the terminal differentiation process are intracellular proteases that cleave epidermal proteins leading to destruction of organelles and formation of the stratum corneum. The overall goal of this project is to determine the function of caspase 14, an epidermal-specific cysteine aspartate protease activated during terminal differentiation, and to examine the role of the free pro-filaggrin terminal peptide which is liberated during the proteolytic processing of the calcium binding protein profilaggrin. Hypotheses to be tested are (1) that caspase-14 plays important biological roles in the keratinization process by cleaving structural and/or regulatory proteins that are critical for initiation and/or execution of terminal differentiation; and (2) that the free profilaggrin terminal, by interacting with other keratinocyte proteins including members of the annexin and 14-3-3 family, has a specific role in regulating profilaggrin processing and other calcium-dependent cytoplasmic or nuclear events that are essential for keratinization. To address these questions, the specific aims proposed are (1) to express caspase-14 in E. coli and purify the active enzyme; (2) to determine the substrate specificity and natural keratinocyte targets of caspase-14; (3) To determine the substrate specificity and natural keratinocyte targets of caspase-14; (3) to determine the function of caspase-14 in vivo by targeted disruption in mice; and (4) to identify proteins that bind the free pro-filaggrin terminal peptide and determine how these interactions affect its intracellular distribution and possible biological function(s). These studies will provide insight into both the biology of epidermal differentiation and the molecular basis of autosomal dominant and recessive ichthyosis such as ichthyosis vulgaris and lamellar ichthyosis that display defects in stratum corneum structure and function of the epidermal barrier.